This project will investigate the molecular mechanism by which a major, well-characterized developmental regulatory hierarchy of Drosophila melanogaster directs the sex-specificity of target gene transcription. The significance of the project lies in its advanced analysis of the structure and function of a cellular enhancer from a higher organism and in its study of the direct molecular connection between a developmental regulatory hierarchy and target genes. Understanding developmental regulation and cellular enhancer function is crucial to understanding defects in gene expression that lead to human disease and human developmental abnormalities. The project will investigate a particular enhancer that conveys the male- specific repression signal from the hierarchy to a pair of target genes. We will determine how this repression signal is received by the enhancer and then sent to the promoters. A gene at the bottom of the regulatory hierarchy encodes a male-specific repressor of female genes and a female- specific repressor of male genes. In vitro both male and female repressors bind to the male-specific regulatory elements of the enhancer. We will examine the mechanism operating in vivo to determine whether the male repressor is sufficient to cause repression in males and whether the female repressor does not bind these elements, or does bind them but does not repress. This will be done by using in vitro mutagenesis/germline transformation methods to identify the functional elements within the enhancer and to determine whether the bound repressor acts on other enhancer elements or directly on the promoter. These studies will show whether this repressor action occurs by steric hindrance or DNA looping mechanisms. We will also investigate the biochemistry of the interactions between the DNA sites and the purified, expressed, repressors. This biochemical portion of the project aims at an explanation for the male and female repressor activities at this male-specific enhancer. We will then use a transcription system derived from an appropriate purified tissue in order to reproduce this sex-specific repression in vitro and investigate the function of proteins that operate in the repression mechanism.